Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii

Abstract

Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta-and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes.

Production optimization of a heat-tolerant alkaline pectinase from Bacillus subtilis ZGL14 and its purification and characterization

“…8,[16][17][18][19][20][21] Based on the pH requirement for optimal enzymatic activity, pectinase can be broadly classified into acidic and alkaline pectinase. [22][23][24][25] Acidic pectinase has been extensively reported for the extraction and clarification of fruit juices and wines, 10,[26][27][28] the maceration of vegetables and fruits, the improvement of essential oil extraction and the enhancement of baby-food production, 18,29 whereas alkaline pectinase is used for pretreating waste water from the food processing industry that contains pectinaceous waste 9 and in the processing and degumming of bast fibers such as ramie (Boehmeria nivea), 8 sunn hemp (Crotalaria juncea), 8 buel (Grewia optiva) 12 and jute (Chorchorus capsularis). 30 It is also widely used in the fabric industry for the retting of plant fibers such as flax, hemp and jute, the biopreparation of cotton fabrics and the enzymatic polishing of jute/cotton blended fabrics; in the paper industry to solve the retention problem in mechanical pulp bleaching and in the treatment of pulp and paper mill effluents; and for improving the quality of black tea.…”

Purification and Characterization of Polygalacturonase Produced by Aspergillus niger AN07 in Solid State Fermentation

“…Purification and characterization of polygalacturonase from several fungal sources, such as A. awamori [14,15], Sclerotium rolfsii [18], Penicillium sp. [20], Bispora sp.…”

“…Mucor flavus [2], M. circinelloides [10], A. niger [11][12][13], A. awamori [14,15], A. carbonarius [16], Rhizopus oryzae ______________________________ *Corresponding author: Email: ak_sbt@yahoo.com [17], Sclerotium rolfsii [18], Penicillium sp. [19,20], Bispora sp.…”

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

“…Thermophilic fungi Thielavia arenaria XZ7 (60°C) (7), Thermoascus aurantiacus CBMAI-756 (60 to 65°C) (11), Penicillium sp. strain SPC-F 20 (60°C) (12), and Sclerotium rolfsii (60°C) (13) have been identified as the most excellent microbial sources of thermophilic or thermostable PGs. There have been diverse protein engineering strategies employed.…”